Presence of a threshold for promoting effects of phenobarbital on diethylnitrosamine-induced hepatic foci in the rat

The dose dependence of the hepatopromoting effects of phenobarbital (PB) was investigated in a rat liver medium-term bioassay (Ito test) to elucidate a practical threshold level. F344 rats were given a single i.p. injection of diethylnitrosamine (200 mg/kg body wt) and subjected to two-thirds partial hepatectomy at week 3. Commencing 2 weeks from the start, PB at doses of 0, 1, 2, 4, 7.5, 15 or 500 p.p.m. in experiment 1 and 0, 0.01, 0.1 or 0.5 p.p.m. in experiment 2 were fed to the rats for 6 weeks. Experiment 3 was conducted to confirm previous data using the same medium-term bioassay, with PB at doses of 0, 1, 2, 4, 7.5, 15, 30, 60, 125, 250 or 500 p.p.m. fed to the rats. All surviving animals were killed at week 8 in these experiments and their livers were immunohistochemically examined for expression of glutathione S-transferase placental form (GST-P). Quantitative values for GST-P-positive foci in the liver were increased dose dependently in rats given 60-500 p.p.m. PB. However, those for doses in the range 1- 7.5 p.p.m. demonstrated a decrease as compared with the control group (0 p.p.m.), with significant differences observed for 1 and 2 p.p.m.. The results for 15-30 and 0.01-0.5 p.p.m. were comparable with the control values. Examination of transforming growth factor-alpha (TGF- alpha)-positive foci also produced similar results to those for GST-P in experiment 1. Immunohistochemical staining of TGF-alpha and GST-P using serial liver sections demonstrated that the TGF-alpha-positive foci comprised a sub-population of the GST-P-positive lesions, being approximately 1/8-1/10th as common in livers of animals treated with PB. TGF-alpha-positive foci were almost always negative on immunostaining for TGF-beta. Western blotting for proteins CYP2B1, 2C6 and 3A2 revealed a good correlation between changes in GST-P-positive foci and CYP3A2 protein expression. The finding of inhibition effects at low doses of PB confirms the presence of a threshold level for promoting effects by PB on liver carcinogenesis in rats.      

Down-regulation of 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx)-induced CYP1A2 Expression Is Associated with Bovine Lactoferrin Inhibition of MeIQx-induced Liver and Colon Carcinogenesis in Rats

The inhibitory influence of bovine lactoferrin (bLF) on induction of preneoplastic hepatic glutathione S -transferase placental form-positive (GST-P⁺) cell foci and colon aberrant crypt foci (ACF) by diethylnitrosamine (DEN) and 2-amino-3,8-dimethylimidazo[4,5- f ]quinoxaline (MeIQx) was investigated in F344 rats. Rats were initially treated with DEN, then placed on basal diet containing MeIQx (200 ppm) alone, MeIQx plus 2% bLF, or MeIQx plus 0.2% bLF from week 2 to week 8, with partial hepatectomy performed at week 3. Concomitant administration of 2% or 0.2% bLF with MeIQx caused significant dose-dependent decreases in both number and unit area of GST-P⁺ cell foci (2% bLF, P <0.001; 0.2% bLF, P <0.01). Similar results were observed for MeIQx-induced colon ACF in the groups without DEN treatment (2% and 0.2% bLF, P <0.05). To investigate the underlying mechanisms, we analyzed the influence of bLF on levels of cytochrome P4501A2 (CYP1A2), a metabolically activating enzyme of MeIQx in the liver. The results demonstrated that combined administration of 2% bLF significantly reduced levels of MeIQx-induced CYP1A2 mRNA ( P <0.05) and protein ( P <0.05) to the normal levels, in association with reduced values for MeIQx-DNA adducts ( P <0.05), liver GST-P⁺ cell foci and colon ACF. These results suggest that bLF is a chemopreventive agent for DEN alone or DEN plus MeIQx-induced liver, and MeIQx-induced colon carcinogenesis in rats. One possible mechanism is a normalizing down-regulation of CYP1A2 expression by bLF, with consequent reduction of carcinogen activation and adduct formation.     

Phenobarbital at low dose exerts hormesis in rat hepatocarcinogenesis by reducing oxidative DNA damage,altering cell proliferation,apoptosis and gene expression

Our recent research indicated that phenobarbital (PB) may inhibit the development of N-diethylnitrosamine (DEN)-initiated pre-neoplastic lesions at low doses in a rat liver medium-term bioassay (Ito test), while high doses exhibit promoting activity. This raises the question of whether treatment with low doses of PB might reduce cancer risk. For clarification, male 6-week-old F344 rats were treated with PB at doses of 0, 2, 15 and 500 p.p.m. in the diet for 10 or 33 weeks after initiation of hepatocarcinogenesis with DEN. In a second, short-term experiment, animals were given PB at doses of 2, 4, 15, 60 and 500 p.p.m. for 8 days. Formation of glutathione S-transferase placental form (GST-P) positive foci and liver tumors was inhibited at 2 p.p.m. Generation of oxidative DNA damage marker, 8-hydroxy-2'-deoxyguanosine (8-OHdG), cellular proliferation within the areas of GST-P positive foci and apoptosis in background liver parenchyma were suppressed. Suppression of 8-OHdG formation by PB at low dose might be related to the enhanced mRNA expression of 8-OHdG repair enzyme, oxoguanine glycosylase 1 (Ogg1). Moreover, as detected by cDNA microarray analysis, PB treatment at low dose enhanced mRNA expression of glutamic acid decarboxylase (GAD65), an enzyme involved in the synthesis of gamma-aminobutyric acid (GABA), and suppressed MAP kinase p38 and other intracellular kinases gene expression. On the contrary, when PB was applied at a high dose, GST-P positive foci numbers and areas, tumor multiplicity, hydroxyl radicals and 8-OHdG levels were greatly elevated with the increase in CYP2B1/2 and CYP3A2 mRNA, protein, activity and gene expression of GST, nuclear tyrosine phosphatase, NADPH- cytochrome P-450 reductase and guanine nucleotide binding protein G(O) alpha subunit. These results indicate that PB exhibits hormetic effect on rat hepatocarcinogenesis initiated with DEN by differentially altering cell proliferation, apoptosis and oxidative DNA damage at high and low doses.     

Butylated hydroxytoluene lacks the activity of phenobarbital in enhancing diethylnitrosamine-induced mouse liver carcinogenesis

The effect of the food additive butylated hydroxytoluene (BHT) as an enhancer of liver carcinogenesis in mice was investigated. Liver carcinogenesis was initiated by intraperitoneal injection of diethylnitrosamine (DEN) in male B6C3F1 mice at 100 or 200 mumol/kg body weight once a week for 10 weeks (total exposure 1000 or 2000 mumol/kg body weight). After an exposure-free recovery interval of 4 weeks, groups of mice were fed either basal diet or diets containing either 5000 ppm BHT or 500 ppm phenobarbital (PB), as a positive control, for 24 weeks. Exposure to the initiating doses of DEN alone induced no liver foci at 10 weeks or at 14 weeks after the recovery period, but at termination at 38 weeks, foci and adenomas were present in a dose-related incidence. In the groups given BHT after DEN/recovery, the incidence and the multiplicity of liver foci and adenomas were not different from those in mice given only DEN/recovery, whereas, in the groups given PB after DEN, liver lesions were increased by 1.7-3.0-fold. In conclusion, BHT had no promoting or syncarcinogenic effect on DEN-induced mouse liver carcinogenesis, whereas under the same conditions, PB acted as an enhancer.     

Synergistic effects of low-dose hepatocarcinogens in induction of glutathione S-transferase P-positive foci in the rat liver

The effects of combined administration of hepatocarcinogens at low doses on the development of glutathione S-transferase P-form (GST-P)-positive foci of rat liver were examined utilizing a bioassay model which consists of a single injection of diethylnitrosamine (DEN, 200 mg/kg, ip), two-thirds partial hepatectomy at week 3 and a 6-week administration of test compounds. The chemicals used, 2-acetylaminofluorene (2-AAF), 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB), phenobarbital (PB), thioacetamide (TAA), N-ethyl-N-hydroxyethylnitrosamine (EHEN), benzo[a]pyrene (B[a]P), carbazole, and alpha-hexachlorocyclohexane (alpha-HCH) were incorporated in the diet, except for EHEN which was dissolved in the drinking water, at levels of 1/6 of the doses usually used. The combinations were: I) 2-AAF, 3'-Me-DAB, PB, TAA, EHEN and B[a]P, II) 2-AAF, 3'-Me-DAB and PB, III) TAA, EHEN and B[a]P, IV) 2-AAF, 3'-Me-DAB, carbazole, TAA, EHEN and alpha-HCH, V) 2-AAF, 3'-Me-DAB and carbazole, and VI) TAA, EHEN and alpha-HCH. All combinations, except for II, caused an increase in the area of the foci as evaluated by the ratios of areas in the combined administration groups to the sum totals of 3 or 6 individual data: I) 1.75, II) 0.81, III) 2.01, IV) 3.62, V) 1.34 and VI) 2.91. The non-synergistic effect in combination II might be related to PB induction of hepatic microsomal enzymes leading to enhanced enzymatical detoxification of 2-AAF and 3'-Me-DAB. The present results indicate that exposure to several chemicals of similar organotropism, even at doses lower than the apparent carcinogenic levels, might be critical to the carcinogenic process.     

A NEW MEDIUM-TERM BIOASSAY SYSTEM FOR DETECTION OF ENVIRONMENTAL CARCINOGENS USING DIETHYLNITROSAMINE-INITIATED RAT LIVER FOLLOWED BY D-GALACTOSAMINE TREATMENT AND PARTIAL HEPATECTOMY

The effects of D-galactosamine on induction of preneoplastic glutathione S-transferase placental form positive liver foci were investigated in F344 rats pretreated with diethylnitrosamine (DEN) in an attempt to improve the predictive value of the medium-term bioassay system developed in our laboratory. Two weeks after the initial single ip dose (200 mg/kg) of DEN, administration of test compounds was commenced simultaneously with an ip injection of D-galactosamine at a dose of 300 mg/kg body wt. All rats were then subjected to two-thirds partial hepatectomy (PH) at week 5 and sacrificed for assessment of lesion yield at week 8. Measurement and comparison of the numbers and areas of glutathione S-transferase placental form positive (GST-P⁺) foci per cm² revealed a positive response to more carcinogens, including non-hepatocarcinogens, than did the same bioassay system without injection of D-galactosamine. Thus the results suggest that inclusion of this extra proliferative stimulus may improve the medium-term detection of carcinogens and modifiers.     

Dose-dependent effects of 2-acetylaminofluorene on hepatic foci development and cell proliferation in rats

Dose-dependent development of pre-neoplastic liver cell foci induced by 2-acetylaminofluorene (2-AAF) was investigated in relation to cell-proliferative activity. Male F344 rats were initially given a single i.p. injection of diethylnitrosamine (DEN, 200 mg/kg) and starting 2 weeks later received diets containing 2-AAF at dose levels of 150, 100, 60, 45, 35 or 30 p.p.m., 500 p.p.m. phenobarbital (PB) or basal diet as a control for 6 weeks. Two-thirds partial hepatectomy (PH) was performed at week 3. The rats were sequentially killed from weeks 0 to 16 and liver sections were analysed by double staining for both BrdU incorporation and glutathione S-transferase placental form (GST-P) expression. 2-AAF increased numbers and areas of GST-P positive (GST-P+) foci in a dose-dependent manner, especially after PH. Proliferation of hepatocytes, as indicated by BrdU labelling indices (LI), was higher in GST-P+ foci than in surrounding hepatocytes in all 2-AAF-treated groups, even after cessation of carcinogen administration. Proliferative response of hepatocytes to PH was delayed in rats treated with the highest dose of 2-AAF in both foci and in surrounding areas possibly due to the 2-AAF toxicity. In the PB treated group, the results were similar to those for the lower dose 2-AAF-treated groups. It is concluded that the development of GST-P+ foci and cell proliferation in GST-P+ foci are directly related to 2-AAF treatment in a dose-dependent manner and the present assay system is reliable for detection of carcinogenicity of chemicals even at low doses.     

Dissimilar patterns of promotion by di(2-ethylhexyl)phthalate and phenobarbital of hepatocellular neoplasia initiated by diethylnitrosamine in B6C3F1 mice

Potentially preneoplastic hepatocellular hyperplastic foci andhepatocellular neoplasms were studied in weanling male B6C3F1mice that received a single i.p. injection (80 mg/kg) of diethylnitrosamine(DEN) at 4 weeks of age, followed by oral administration ofphenobarbital (PB) or di(2-ethylhexyl)- phthalate (DEHP) thatbegan 2 weeks after DEN injection and continued for up to 6months. PB was administered in drinking water at 500 p.p.m.and DEHP in the feed at 3000, 6000 or 12 000 p.p.m. Groups ofmice were sacrificed at 2, 4 and 6 months after DEN exposure;formalin-fixed liver samples were evaluated histologically.Hepatocellular neoplasms and foci of hyperplasia were quantifiedwith the aid of an image analysis computer. Few foci were seenat 2, 4 or 6 months in mice exposed to DEN, PB or DEHP alone,while numerous foci and neoplasms were seen in mice given DEHPor PB after DEN. Area-perimeter measurements for each hepatocellularfocus or neoplasm transection revealed that foci and neoplasmsin PB-exposed mice increased both in size (area and volume)and in number throughout the study. In DEHP-exposed mice thepattern of response was different in that the numbers of focidid not increase between 4 and 6 months, but the foci increasedin mean diameter and volume throughout the experiment. Fociand tumors appeared earlier in mice given higher dietary levelsof DEHP than in those given lower doses. By the end of the studythe number of foci per unit volume of liver was similar in micegiven any dose of DEHP, but their volume was dose-related. Hepatocellularfoci and neoplasms in PB-exposed mice were composed predominantlyof eosinophilic hepatocytes, while in DEHP exposed mice, basophilicfoci and neoplasms predominated; the latter were more malignantin appearance than neoplasms in PB-exposed mice. At 6 months,the neoplasms in high dose DEHP-exposed mice were significantlylarger than those in PB exposed mice. Histochemistry, however,revealed similarities between lesions in mice exposed to PBor DEHP. PB given continuously for 6 months revealed no initiatingactivity of DEHP given once by gavage and followed by PB indrinking water. Both morphology and biology of hepatocellularfoci and neoplasms, which develop in mice after a single exposureto a carcinogen with initiating activity, thus depend, in part,on the subsequent promoting agent. More than one process oftumor promotion, as characterized by a specific sequence ofmorphologic and biochemical changes, is possible for the mousehepatocyle.     

Different responses to phenobarbital promotion in the development of gamma-glutamyltranspeptidase-positive foci in the liver of rats initiated with diethylnitrosamine, N-hydroxy-2-acetyl-aminofluorene and aflatoxin B1

The promoting activities of phenobarbital (PB) on the development of gamma-glutamyl-transpeptidase-positive (gamma-GT+) foci in rat liver with three different initiating agents were compared in a short-term system (8 weeks). Male F344 rats were initiated by a single application of 200 mg/kg of diethylnitrosamine (DEN), 30 mg/kg of N-hydroxy-2-acetylaminofluorene (N-OH-AAF), 1.0 or 0.5 mg/kg of aflatoxin B1 (AFB1) or the vehicles alone. Two weeks after the initiation, animals were placed on a 0.05% PB diet for 6 weeks. Partial hepatectomy was performed at the end of the third week of the experiment. As a positive control, some animals were fed diet containing 0.06% 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) after the initiation. The number and area of gamma-GT+ foci in the liver were quantified. All three initiators showed a summation effect with 3'-Me-DAB on the appearance of gamma-GT+ foci. Promotion by PB, however, was observed only in DEN-initiated rats and not in N-OH-AAF- or AFB1-initiated rats. It is apparent from the present experimental data that the promoting potential of PB on liver carcinogenesis depends on the initiating agent.     

Expression of cytochrome P450 2A5 in preneoplastic and neoplastic mouse liver lesions

Cytochrome P450 (CYP) 2A5 is involved in the metabolism of carcinogens like aflatoxin B₁ and N-nitrosodiethylamine (NDEA), and CYP2A5 levels are increased in some pathological states of the liver (e.g., infectious hepatitis and porphyria). We analyzed the expression of CYP2A5 during experimental liver carcinogenesis in three different mouse strains (C3H/He, C57BL/6J, and B6C3F1) with immunohistochemical techniques and in situ hybridization. In normal liver, CYP2A5 protein and mRNA were detected in centrilobular hepatocytes only. Phenobarbital treatment increased the number of CYP2A5-positive centrilobular hepatocytes and the CYP2A5-positive areas were extended into the middle zone in all strains, but periportal hepatocytes remained negative. Fifty percent of the spontaneous foci in untreated mice, over 90% of the foci in mice treated with NDEA or phenobarbital and all of the hepatocellular adenomas and carcinomas displayed positive immunostaining and a strong CYP2A5 mRNA signal by in situ hybridization. In the liver tumors metastasized to the lung, expression of CYP2A5 had largely disappeared. CYP2A5 expression in neoplastic and putative preneoplastic lesions, although sometimes heterogeneous, was apparently independent of the typical zonal expression pattern in normal tissue. As expected, the C57BL/6J mice developed fewer foci and tumors than the C3H/He and B6C3F1 mice, but the phenotype of CYP2A5 overexpression was similar in all the strains. Our data suggest that the increased expression of CYP2A5 may play an important role in the development of liver cancer in mice and may be used as a novel marker for spontaneous and NDEA-induced mouse liver foci. Mol. Carcinog. 22:229–234, 1998. © 1998 Wiley-Liss, Inc.     

Alpha-benzene hexachloride exerts hormesis in preneoplastic lesion formation of rat hepatocarcinogenesis with the possible role for hepatic detoxifying enzymes

Recently there has been a shift in the prevailing paradigm regarding the dose dependence of carcinogen action with increasing acceptance of hormesis phenomenon, although underlying mechanisms remain to be established. To ascertain whether alpha-benzene hexachloride (alpha-BHC) might act by hormesis, rats were initiated with diethylnitrosamine and then alpha-BHC ranging from 0.01 to 500 ppm was administered in the diet for 10 weeks. The highest concentration of alpha-BHC significantly increased the number and area of glutathione S-transferase placental form (GST-P) positive foci, preneoplastic lesions in the liver, but its low dose, 0.05 ppm, caused significant reduction, showing a J-shape dose-response curve. The proliferating cell nuclear antigen positive index for GST-P positive foci in the low dose-treated group was significantly reduced. The dose response curves of CYP450 content, NADPH-P450 reductase activity and 8-hydroxydeoxyguanosine formation revealed the same pattern as GST-P positive foci data. The response curves of CYP2B1 and 3A2 in their activities, protein and mRNA expression showed a threshold but CYP2C11 activity exhibited an inverted J-shape. These results might suggest the possibility of hormesis of alpha-BHC at early stages of rat hepatocarcinogenesis. The possible mechanism involves induction of detoxifying enzymes at low dose, influencing free radical production and oxidative stress, and consequently pathological change in the liver.     

Promoting effects of monomethylarsonic acid,dimethylarsinic acid and trimethylarsine oxide on induction of rat liver preneoplastic glutathione S-transferase placental form positive foci: a possible reactive oxygen species mechanism

Dimethylarsinic acid (DMA) is a major metabolite of inorganic arsenicals, which are epidemiologically significant chemicals in relation to liver cancer in mammals. The present study was conducted to determine the promoting effects of organic arsenicals related to DMA [monomethylarsonic acid (MMA) and trimethylarsine oxide (TMAO)] on rat liver carcinogenesis using a liver medium-term bioassay (the Ito test). Male, 10-week-old, F344 rats were given a single i.p. injection of diethylnitrosamine at a dose of 200 mg/kg b.w. as an initiator. Starting 2 weeks thereafter they received 100 ppm of MMA, DMA or TMAO in their drinking water, or no supplement as a control, for 6 weeks. All animals underwent 2/3 partial hepatectomy in week 3 after initiation. Quantification of glutathione S-transferase placental form (GST-P)-positive foci as preneoplastic lesions in liver sections revealed significantly increased numbers and areas in all 3 treated groups compared with controls. Hepatic microsome cytochrome P-450 content was markedly increased with all 3 arsenic treatments. Markedly elevated CYP 2B1 protein levels and CYP 2B1/2 mRNA levels were thus observed in all cases. The potency of promotion was similar for MMA, DMA and TMAO. Since hydroxyradicals were found to be generated in the relatively early phase while methylated arsenicals were metabolized in liver, the resultant oxidative stress might have promoted lesion development.     

Inhibitory Effect of Chlorophyllin on Diethylnitrosamine and Phenobarbital-induccd Hepatocarcinogenesis in Male F344 Rats

Modifying effects of chlorophyllin (CHL) on the diethylnitrosamine (DEN)-phenobarbital (PB) hepatocarcinogenesis model were examined in rats. Five-week-old male F344 rats were divided into 8 groups. Groups 1 through 5 were given i.p. injections of DEN (100 mg/kg body weight) once a week for 3 weeks beginning one week after the start of the experiment, while groups 6 through 8 received vehicle treatment. Groups 1, 2, 3 and 7 received drinking water with 500 ppm PB from one week after the end of carcinogen or vehicle treatment. CHL-containing diet (2000 ppm) was given to group 2 during the initiation phase and to groups 3 and 5 during the promotion and the post-initiation phase, respectively. Group 6 was given the experimental diet alone throughout the experiment (24 weeks). Liver neoplasms were present in DEN-treated groups and PB treatment promoted liver tumorigenesis. The incidences of adenoma in groups 2 and 3 were significantly smaller than in group 1 (P<0.05 and P<0.02), although the reductions in the incidences of liver cell cancer were not significant. The average numbers of liver neoplasms/rat in group 2 were significantly smaller than in group 1 (P<0.05-P<0.005), Glutathione S -transferase placental form-positive foci were also significantly decreased by CHL treatment (P<0.05 and P<0.001). DEN and PB exposure increased liver ornithine decarboxylase activity and this increase was significantly inhibited by feeding of CHL during the initiation phase (P<0.001). These results suggest that CHL is a chemopreventive agent for liver neoplasia.     

Presence of a no-observed effect level for enhancing effects of development of the alpha-isomer of benzene hexachloride (alpha-BHC) on diethylnitrosamine-initiated hepatic foci in rats

The dose dependence of the promoting effects of the alpha-isomer of benzene hexachloride (alpha-BHC) on hepatocarcinogenesis was investigated in a medium-term rat liver bioassay (Ito test). A total of 195 F344 male rats, 6 weeks old, were given a single intraperitoneal injection of diethylnitrosamine (DEN) at the start of the experiment and subjected to two-thirds partial hepatectomy at week 3. Two weeks after the administration of DEN, alpha-BHC were fed to rats at doses of 0, 0.01, 0.1, 0.5, 1, 2, 4, 7.5, 15, 30, 60, 125 and 500 ppm in diet for 6 weeks. All surviving animals were killed at week 8, and their livers were examined immunohistochemically for detection of glutathione S-transferase placental form (GST-P)-positive foci, surrogate preneoplastic lesions. Quantitative values for numbers and areas were dose-dependently increased in rats given alpha-BHC at 0.5-500 ppm. However, those for groups treated with 0.01 and 0.1 ppm were decreased, albeit not significantly in comparison to the controls. Cytochrome P450 3A2 (CYP3A2) protein levels and activities showed a good correlation to the number and area of GST-P-positive foci. These results support evidence of hormesis and indicate a no-observed effect level for alpha-BHC promoting potentials may exist regarding rat liver carcinogenesis, which correlates with expression of CYP3A2 in the liver.     

Synergistic Effects of Low-dose Hepatocarcinogens in Induction of Glutathione S-Transferase P-positive Foci in the Rat Liver

The effects of combined administration of hepatocarcinogens at low doses on the development of glutathione S -transferase P-form (GST-P)-positive foci of rat liver were examined utilizing a bioassay model which consists of a single injection of diethylnitrosamine (DEN, 200 mg/kg, ip), two-thirds partial hepatectomy at week 3 and a 6-week administration of test compounds. The chemicals used, 2-acetylaminofluorene (2-AAF), 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB), phenobarbital (PB), thioacetamide (TAA), N-ethyl-N-hydroxyethylnitrosamine (EHEN), benzo[ a ]pyrene (B[a]P), carbazole, and α-hexachlorocyclohexane (α-HCH) were incorporated in the diet, except for EHEN which was dissolved in the drinking water, at levels of 1/6 of the doses usually used. The combinations were: I) 2-AAF, 3'-Me-DAB, PB, TAA, EHEN and B[a]P, II) 2-AAF, 3'-Me-DAB and PB, III) TAA, EHEN and B[a]P, IV) 2-AAF, 3'-Me-DAB, carbazole, TAA, EHEN and a-HCH, V) 2-AAF, 3'-Me-DAB and carbazole, and VI) TAA, EHEN and α-HCH. All combinations, except for II, caused an increase in the area of the foci as evaluated by the ratios of areas in the combined administration groups to the sum totals of 3 or 6 individual data: I) 1.75, II) 0.81, III) 2.01, IV) 3.62, V) 1.34 and VI) 2.91. The non-synergistic effect in combination II might be related to PB induction of hepatic microsomal enzymes leading to enhanced enzymatical detoxification of 2-AAF and 3'-Me-DAB. The present results indicate that exposure to several chemicals of similar organotropism, even at doses lower than the apparent carcinogenic levels, might be critical to the carcinogenic process.     

Inhibition of 2-amino-3-methylimidazo[4,5-f]quinoline-DNA adducts by indole-3-carbinol: dose-response studies in the rat colon

Indole-3-carbinol (I3C) inhibits the formation of colonic aberrant crypt foci and DNA adducts in rats given heterocyclic amine colon carcinogens, such as 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). Mechanism studies indicate that I3C induces cytochromes P4501A1 and 1A2 (CYP1A1 and CYP1A2), isozymes that respectively metabolize IQ via ring hydroxylation or activate the carcinogen by N-hydroxylation. The present study examined the dose-response for induction of CYP1A1 versus CYP1A2 by I3C, and compared the profiles of induction with the dose- response for inhibition of IQ-DNA adducts in the colon of the F344 rat. Dietary equivalent doses of I3C in the range 100-1000 p.p.m. increased in a dose-related manner both ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) activities in the liver and colonic mucosa, and Western blots showed a corresponding induction of CYP1A1 and CYP1A2 proteins. However, dietary equivalent doses of I3C in the range 10-25 p.p.m. (i) reduced hepatic EROD and MROD activities and CYP1A protein levels compared with controls, (ii) increased the ratio of CYP1A2 versus CYP1A1, and (iii) activated IQ to a more potent mutagen when liver microsomes from rats given I3C were used for metabolic activation in the Salmonella assay. Rats given a single oral dose of I3C shortly before administering IQ (5 mg/kg body wt, p.o.) exhibited dose-related inhibition of colonic IQ-DNA adducts in the range 25-100 p.p.m. I3C, reaching 95% inhibition at doses > or = 100 p.p.m. I3C, but IQ-DNA adducts were elevated slightly at the lowest I3C dose as compared with the controls. The possible significance of the low versus high dose effects of I3C are discussed in the context of human dietary exposures to I3C and the reported chemopreventive mechanisms of I3C in vivo.      

Chemopreventive Effects of Taurine on Diethylnitrosamine and Phenobarbitalinduced Hepatocarcinogenesis in Male F344 Rats

Modifying effects of taurine, a naturally occurring organosulfur compound, on diethylnitrosamine (DEN) and phenobarbital (PB)-induced hepatocarcinogenesis were examined in rats. Male F344 rats, 5 weeks old, were divided into 8 groups. Rats of groups 1 through 5 were given i.p. injections of DEN (100 mgAg body weight) once a week for 3 weeks from one week after the start of the experiment. Of them, animals of group 2 received taurine mixed in a basal diet at a concentration of 2000 ppm for the initial 4 weeks, and those of groups 3 and 5 were given the agent starting 4 weeks after the beginning of experiment until the end (24 weeks). Rats in groups 1, 4, 7 and 8 were kept on the basal diet throughout the experiment (24 weeks). Group 6 was given taurine throughout the experiment and group 8 was treated as a vehicle control. Animals of groups 1,2, 3 and 7 received PB in drinking water at a dose of 500 ppm from one week after the end of carcinogen or vehicle treatment. Liver neoplasms were recognized only in DEN-treated groups. The incidence and average number of liver neoplasms of group 3 were significantly lower than those of group 1. The number of glutathione S-transferase placenta! form (GST-P)-positive foci of group 2 or 3 was significantly smaller than that of group 1 ( P < 0.01 or P < 0,005). The average and unit areas of GST-P-positive foci in groups 2 and 3 were also significantly smaller than those in group 1 ( P < 0.005 and P < 0.0001, P < 0.0001 and P < 0.001, respectively). In this study, the level of ornithine decarboxylase activity in non-neoplastic liver tissue was reduced by taurine treatment in both the initiation and postinitiation phases. These results suggest that taurine could be a chemopreventive agent for liver neoplasia.     

Effect of carcinogen dose fractionation, diet and source of F344 rat on the induction of colonic aberrant crypts by 2-amino-3-methylimidazo[4,5-f]quinoline

Carcinogen dose fractionation, diet and source of laboratory animal were examined as variables in the induction of colonic aberrant crypt foci (ACF) by the heterocyclic amine 2-amino-3-methylimidazo [4, 5-f]quinoline (IQ). In the first experiment, male F344 rats from the National Cancer Institute (NCI rats) were fed AIN-93G diet and, starting in the third week, IQ was given by gavage on alternating days, the total carcinogen dose of 105 mg being fractionated proportionally over 2, 4, 8 or 14 weeks. Only the high dose (2 week) treatment with IQ was effective for the induction of ACF at 16 weeks, producing on average 3.8 ACF/colon versus 0.5 ACF/colon in all other groups (P < 0.05). The 2 week IQ dosing protocol was used in a second experiment in which male F344 rats from Simonsen Laboratories (SN) or NCI were fed AIN-93G, AIN-76A or chow diet. On average, SN rats on chow diet had twice the number of aberrant crypts compared with NCI rats given the same diet and three to four times as many aberrant crypts as NCI rats fed AIN diets. Hepatic cytochrome P4501A1 (CYP1A1) levels were essentially unaffected by diet, but methoxyresorufin O-demethylase activities and CYP1A2 protein levels were increased 2- to 3-fold in animals fed chow versus AIN diets. During the 2 week period of carcinogen administration, IQ markedly induced CYP1A proteins and negated the differences among groups related to diet. No consistent diet-related changes were detected in the activities of aryl sulfotransferase or N-acetyltransferase, but UDP-glucuronosyltransferase activities were elevated 2- to 3-fold in rats given chow versus AIN diets. In summary, high dose treatment with IQ was required for the induction of ACF, rats on the chow diet had more aberrant crypts than those given AIN diets and male F344 rats purchased from different vendors and fed chow diet differed with respect to their sensitivity to induction of ACF.     

单剂量黄曲霉毒素B1致大鼠肝癌作用的短期实验模型

本文报告单剂量黄曲霉毒素Ｂ１（ＡＦＢ１）致大鼠肝癌作用短期实验模型的研究。６周龄、雄性Ｗｉｓｔａｒ大鼠，经腹腔一次性注射不同剂量（０、０．５０、０．７５、１．００和１．５０ｍｇ／ｋｇ体重）的ＡＦＢ１，作为启动剂，两周后，饲以含０．０１５％的２－乙酰氨基芴（２－ＡＡＦ）饲料４周，实验第三周末，施行肝大部分切除术（ＰＨ）。所有动物于实验第６周处死，取肝组织作Ｇａｍｍａ－谷氨酰转肽酶（ＧＧＴ）化学染色，     

Promoting effects of 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene in mouse hepatocarcinogenesis

The effects of phenobarbital (PB) and 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene(TCPOBOP) on liver hyperplasia, induction of microsomal enzymeactivities, and two-stage hepatocarcinogenesis were evaluatedin B6C3F1 female mice. For 4 weeks four groups of mice receivedPB (500 p.p.m. in the drinking water), TCPOBOP (3 mg/kg i.p.once every week), PB together with TCPOBOP or corn oil vehiclei.p. TCPOBOP induced liver hyperplasia and hypertrophy and increasedp-nitroanisole-O-demethylase and aminopyrine-N-demethylase morethan PB. Neither chemical changed UDPG-transferase activity.The association of PB and TCPOBOP gave the same effects as TCPOBOPalone. Other four groups of mice were treated with N-nitroso-N-diethylamineat 7 days of age and then, starting from 8 weeks of age, receivedthe above specified weekly treatments for 20 weeks and werethen sacrificed. Hepatocellular nodules > 150 µm werefound in all animals of all groups. Due to increased size ofthe liver compared to controls, the number of nodules/cm³ decreasedafter PB and TCPOBOP treatments given alone or together; howeverthe mean volume of nodules and the percentage of liver volumeoccupied by nodules increased after TCPOBOP but not after BPtreatment, and the association of PB and TCPOBOP was even moreeffective than TCPOBOP alone. Hepatocellular adenomas >2.4mm in diameter were observed in 5 of 10 TCPOBOP-treated mice(total of 11 nodules) and in 5 of 11 mice that received PB plusTCPOBOP (total of 15 nodules). Hepatocellular carcinomas wereseen in one mouse treated with PB and in three mice given PBand TCPOBOP.